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Biomedical subjects

B O'Rourke

Publications and source records attributed to B O'Rourke.

At least 55 records · Page 3Linked to original sources
PubMed1996

Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure.

Membrane current abnormalities have been described in human heart failure. To determine whether similar current changes are observed in a large animal model of heart failure, we studied dogs with pacing-induced cardiomyopathy. Myocytes isolated from the midmyocardium of 13 dogs with heart failure induced by 3 to 4 weeks of rapid ventricular pacing and from 16 nonpaced control dogs did not differ in cell surface area or resting membrane potential. Nevertheless, action potential duration (APD) was significantly prolonged in myocytes isolated from failing ventricles (APD at 90% repolarization, 1097 +/- 73 milliseconds [failing hearts, n = 30] versus 842 +/- 56 milliseconds [control hearts, n = 25]; P < .05), and the prominent repolarizing notch in phase 1 was dramatically attenuated. Basal L-type Ca2+ current and whole-cell Na+ current did not differ in cells from failing and from control hearts, but significant differences in K+ currents were observed. The density of the inward rectifier K+ current (IKl) was reduced in cells from failing hearts at test potentials below -90 mV (at -150 mV, -19.1 +/- 2.2 pA/pF [failing hearts, n = 18] versus -32.2 +/- 5.1 pA/pF [control hearts, n = 15]; P < .05). The small outward current component of IKl was also reduced in cells from failing hearts (at -60 mV, 1.7 +/- 0.2 pA/pF [failing hearts] versus 2.5 +/- 0.2 pA/pF [control hearts]; P < .05). The peak of the Ca(2+)-independent transient outward current (Ito) was dramatically reduced in myocytes isolated from failing hearts compared with nonfailing control hearts (at +80 mV, 7.0 +/- 0.9 pA/pF [failing hearts, n = 20] versus 20.4 +/- 3.2 pA/pF [control hearts, n = 15]; P < .001), while the steady state component was unchanged. There were no significant differences in Ito kinetics or single-channel conductance. A reduction in the number of functional Ito channels was demonstrated by nonstationary fluctuation analysis (0.4 +/- 0.03 channels per square micrometer [failing hearts, n = 5] versus 1.2 +/- 0.1 channels per square micrometer [control hearts, n = 3]; P < .001). Pharmacological reduction of Ito by 4-aminopyridine in control myocytes decreased the notch amplitude and prolonged the APD. Current clamp-release experiments in which current was injected for 8 milliseconds to reproduce the notch sufficed to shorten the APD significantly in cells from failing hearts. These data support the hypothesis that downregulation of Ito in pacing-induced heart failure is at least partially responsible for the action potential prolongation. Because the repolarization abnormalities mimic those in cells isolated from failing human ventricular myocardium, canine pacing-induced cardiomyopathy may provide insights into the development of repolarization abnormalities and the mechanisms of sudden death in patients with heart failure.

Action Potentials↗
PubMed1996

Synergistic modulation of ATP-sensitive K+ currents by protein kinase C and adenosine. Implications for ischemic preconditioning.

Ischemic preconditioning has been shown to involve the activation of adenosine receptors, protein kinase C (PKC), and ATP-sensitive K+ (K ATP) channels. We investigated the effects of PKC activation and adenosine on K(ATP) current (I KATP) and action potentials in isolated rabbit ventricular myocytes. Responses to pinacidil (100 to 400 micromol/L), an opener of K(ATP) channels, were markedly increased by preexposure to the PKC activator phorbol 12-myristate 13-acetate (PMA, 100 nmol/L). I(KATP) measured at 0 mV was increased by PMA pretreatment from 0.55 +/- 0.32 to 3.25 +/- 0.47 nA (n=6, P < .01). We next determined whether PKC activation abbreviates the time required to turn on I(KATP) developed after an average of 15.1 +/- 2.4 minutes (n=8). Ten-minute pretreatment with PMA alone (PMA+MI) did not significantly alter this latency (11.9 +/- 2.0 minutes, n=8). Since adenosine receptor activation has been shown to play an important role in the preconditioning response, two groups of myocytes were studied with adenosine (10 micromol/L) included during MI. Without PMA, adenosine alone (MI+Ado) did not affect the latency to develop I(KATP) (12.3 +/- 1.5 minutes, n=8). However, if cells were pretreated with PMA and then subjected to MI in the presence of adenosine (PMA+MI+Ado), the latency was greatly shortened to 5.5 +/- 1.6 minutes (n=8;P < .02 versus MI, PMA+MI, and MI+Ado groups). This effect could not be reproduced by an inactive phorbol but was completely abolished by the adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline. The opening of K(ATP) channels may be cardioprotective because of the abbreviation of action potential duration (APD) during ischemia. Therefore, we tested whether PKC activation could modify the time course of APD shortening during MI. Consistent with the ionic current measurements, PMA pretreatment significantly accelerated APD shortening, but only when adenosine (10 micromol/L) was included during MI. The effects were not attributable to accelerated ATP consumption: PMA pretreatment did not alter the time required to induce rigor during MI, whether or not adenosine was included. Our results indicate that PKC activation increases the I(KATP) Induced by pinacidil or by MI. The latter effect requires concomitant adenosine receptor activation. The synergistic modulation of I(KATP) by PKC and adenosine provides an explicit basis for current paradigms of ischemic preconditioning.

Adenosine↗
PubMed1995

Presynaptic modulation of cortical synaptic activity by calcineurin.

Synaptic plasticity is modulated by Ca(2+)-induced alterations in the balance between phosphorylation and dephosphorylation. Recent evidence suggests that calcineurin, the Ca(2+)-calmodulin-dependent phosphatase (2B), modulates the activity of postsynaptic glutamate receptors. However, in rat cortex, calcineurin is enriched mainly in presynaptic, not postsynaptic, fractions. To determine if calcineurin modulates glutamatergic neurotransmission through a presynaptic mechanism, we used whole-cell patch clamp experiments to test effects of two specific calcineurin inhibitors, cyclosporin A (CsA) and FK506, on synaptic activity in fetal rat cortical neurons. The rate of spontaneous action-potential firing was markedly increased by either CsA or FK506 but was unaffected by rapamycin, a structural analog of FK506 which has no effect on calcineurin. In voltage-clamp experiments, CsA increased the rate but not the amplitude of glutamate receptor-mediated, excitatory postsynaptic currents, suggesting an increased rate of glutamate release. CsA had no effect on the amplitude of currents evoked by brief bath application of selective glutamate receptor agonists, providing further evidence for a pre- rather than postsynaptic site of action. In conclusion, these data indicate that calcineurin modulates glutamatergic neurotransmission in rat cortical neurons through a presynaptic mechanism.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1995

Metabolic oscillations in heart cells.

Oscillatory rhythms underlie biological processes as diverse and fundamental as neuronal firing, secretion, and muscle contraction. We have detected periodic changes in membrane ionic current driven by intrinsic oscillations of energy metabolism in guinea pig heart cells. Withdrawal of exogenous substrates initiated oscillatory activation of ATP-sensitive potassium current and cyclical suppression of depolarization-evoked intracellular calcium transients. The oscillations in membrane current were not driven by pacemaker currents or by alterations in intracellular calcium and thus represent a novel cytoplasmic cardiac oscillator. The linkage to energy metabolism was demonstrated by monitoring oscillations in the oxidation state of pyridine nucleotides. Interventions which altered the rate of glucose metabolism modulated the oscillations, suggesting that the rhythms originated at the level of glycolysis. The metabolic oscillations produced cyclical changes in electrical excitability, underscoring the potential importance of this intrinsic oscillator in the genesis of cardiac arrhythmias.

Animals↗
PubMed1995

Radiographic detection of overhangs formed by resin composite luting agents.

OBJECTIVES: An in vitro model was used to assess the ability of standard radiographic techniques to detect marginal overhangs of resin composite luting agents beside porcelain and resin composite inlays. METHODS: The radiodensity of five commercially available luting resins was determined using ISO 4049 methodology. For four of the luting agents, artificial overhangs (0.5 x 0.5 x 2 mm) were created at the cervical margin of standard resin composite and porcelain inlays. Radiographs were recorded, using wax as a tissue equivalent, and the overhangs reduced incrementally in depth by 0.5 mm with serial images at each depth. The images were assessed in random sequence by three observers. RESULTS: There were significant differences between the radiopacity of the luting resins. These correlated well with the ability of the observers to detect marginal overhangs adjacent to resin composite inlays. CONCLUSIONS: Even with the most radiopaque luting material, a substantial marginal ledge could not be detected in association with a radiopaque resin composite inlay. The threshold for detection of the overhang was lower when using a radiolucent porcelain inlay.

Analysis of Variance↗
PubMed1995

Inward rectifier K+ channel from human heart and brain: cloning and stable expression in a human cell line.

We have cloned the human homologue of the inward rectifier K+ channel from both heart and brain tissue (HHBIRK1). The human clones were identical to each other in their coding regions and were highly homologous to the mouse macrophage (IRK1) channel. The inward rectifier currents from human and mouse clones were characterized using a novel strategy for stable ion channel expression in a human cell line. The permeability of the expressed inwardly rectifying channels was greater for K+ than for Rb+, whereas no current was observed when K+ was replaced by Na+. A prominent time- and voltage-dependent block was observed in the presence of Ba2+, whereas a small decay in the steady-state current was observed with millimolar concentrations of Na+. Single-channel conductances of 49.1 +/- 3.3 pS (n = 6) and 40.2 +/- 2.5 pS (n = 3) (P = 0.005) were obtained for the HHBIRK1 and IRK1 clones, respectively. These results indicate that sequence dissimilarities between human and mouse inward rectifier K+ channels may have significant functional consequences.

Adult↗
PubMed1995

Functional consequences of sulfhydryl modification in the pore-forming subunits of cardiovascular Ca2+ and Na+ channels.

The structure and function of many cysteine-containing proteins critically depend on the oxidation state of the sulfhydryl groups. In such proteins, selective modification of sulfhydryl groups can be used to probe the relation between structure and function. We examined the effects of sulfhydryloxidizing and -reducing agents on the function of the heterologously expressed pore-forming subunits of the cloned rabbit smooth muscle L-type Ca2+ channel and the human cardiac tetrodotoxin-insensitive Na+ channel. The known sequences of the channels suggest the presence of three or four cysteine residues within the putative pores of Ca2+ or Na+ channels, respectively, as well as multiple other cysteines in regions of unknown function. We determined the effects of sulfhydryl modification on Ca2+ and Na+ channel gating and permeation by using the whole-cell and single-channel variants of the patch-clamp technique. Within 10 minutes of exposure to 2,2'-dithiodipyridine (DTDP, a specific lipophilic oxidizer of sulfhydryl groups), Ca2+ current was reduced compared with the control value, with no significant change in the kinetics and no shift in the current-voltage relations. The effect could be readily reversed by 1,4-dithiothreitol (an agent that reduces disulfide bonds). Similar results were obtained by using the hydrophilic sulfhydryl-oxidizing agent thimerosal. The effects were Ca(2+)-channel specific: DTDP induced no changes in expressed human cardiac Na+ current. Single-channel Ba2+ current recordings revealed a reduction in open probability and mean open time by DTDP but no change in single-channel conductance, implying that the reduction of macroscopic Ca2+ current reflects changes in gating and not permeation. In summary, the pore-forming (alpha 1) subunit of the L-type Ca2+ channel contains functionally important free sulfhydryl groups that modulate gating. These free sulfhydryl groups are accessible from the extracellular side by an aqueous pathway.

2,2'-Dipyridyl↗
PubMed1994

Oscillations of membrane current and excitability driven by metabolic oscillations in heart cells.

Periodic changes in membrane ionic current linked to intrinsic oscillations of energy metabolism were identified in guinea pig cardiomyocytes. Metabolic stress initiated cyclical activation of adenosine triphosphate-sensitive potassium current and concomitant suppression of depolarization-evoked intracellular calcium transients. The oscillations in membrane current and excitation-contraction coupling were linked to oscillations in the oxidation state of pyridine nucleotides but were not driven by pacemaker currents or alterations in the concentration of cytosolic calcium. Interventions that altered the rate of glucose metabolism modulated the oscillations, suggesting that the rhythms originated at the level of glycolysis. The energy-driven oscillations in potassium currents produced cyclical changes in the cardiac action potential and thus may contribute to the genesis of arrhythmias during metabolic compromise.

Adenosine Triphosphate↗
PubMed1992

Phosphorylation-independent modulation of L-type calcium channels by magnesium-nucleotide complexes.

Free magnesium ions and magnesium-nucleotide complexes can exert opposite effects on many fundamental cellular processes. Although increases in the intracellular concentration of magnesium ions inhibit the L-type calcium current in heart cells, magnesium-adenosine triphosphate complexes (MgATP) would be expected to increase the current by promoting channel phosphorylation. Rapid increases in the intracellular concentration of MgATP induced by flash photolysis of caged magnesium or caged ATP resulted in enhanced calcium current. The increase in calcium current was not prevented by blocking phosphorylation, revealing a previously unrecognized direct regulatory action of the magnesium-nucleotide complex.

Adenosine Triphosphate↗
PubMed1992

Alpha-adrenergic modification of the Ca2+ transient and contraction in single rat cardiomyocytes.

Intracellular Ca2+ transients and contraction were measured simultaneously in single rat cardiomyocytes loaded with the fluorescent Ca2+ indicator fura-2, using a recently described high-speed digital imaging method (O'Rourke et al., 1990, Am J Physiol 259: H230-H242). In cardiomyocytes electrically-stimulated at 1 Hertz, alpha-adrenoceptor activation in the presence of beta-adrenoceptor blockade resulted in enhanced cell shortening associated with an increase in the amplitude of the cytosolic Ca2+ transient. Both effects developed in parallel over a 10-min time period and occurred without a change in the half-times for decay of Ca2+ or relaxation of the cell. To determine if the increase in contractility was proportional to the increase in peak cytosolic Ca2+, the effect of raising extracellular Ca2+ ([Ca2+]o) from 0.5 to 3 mM was examined in the absence and presence of alpha-adrenoceptor activation. At [Ca2+]o concentrations up to 1 mM, alpha-adrenoceptor-mediated effects on contraction were directly correlated with changes in peak cytosolic Ca2+ and resembled the effect of raising [Ca2+]o alone. In 2 and 3 mM [Ca2+]o, peak cytosolic Ca2+ approached a maximal level and alpha-adrenoceptor activation induced a slight enhancement in the extent of shortening in the absence of a detectable alteration of the Ca2+ transient. In contrast, under similar conditions, beta-adrenergic effects on shortening never exceeded those of alpha-adrenoceptor activation, although much higher peak cytosolic Ca2+ concentrations were achieved at high [Ca2+]o. The results suggest that the mechanism underlying the positive inotropic effect of alpha-adrenergic stimulation in rat ventricular cells is primarily dependent on an enhancement of the cytosolic Ca2+ transient, although there is also an increase in the myofibrillar response to intracellular Ca2+ under the condition of high extracellular Ca2+.

Animals↗
PubMed1992

Trauma triage: a nine-year experience.

STUDY OBJECTIVE: To describe the Orange County trauma system's nine-year (July 1980 through June 1989) experience with patient triage. Changes in patterns were sought after the 1983 revisions in triage criteria. DESIGN: A nine-year retrospective analysis of trends in trauma patient triage in Orange County. A pre-analysis and postanalysis to discern changes in trends after a July 1983 revision in triage criteria was conducted. SETTING: Orange County, California, Emergency Medical Services (EMS) Agency, serving an urban population of more than 2 million. Orange County EMS responds to about 90,000 calls annually, 3,500 of which are for trauma patients. Twenty-eight nontrauma and neurosurgical paramedic receiving centers and four paramedic trauma receiving centers served the community during the study period. TYPE OF PARTICIPANTS: All patients triaged to Orange County trauma centers between July 1980 and June 1989. Single neurologic injury patients were triaged to neurosurgical receiving centers and are not included. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Logarithmic regression analysis showed a 386% increase in the number of trauma patients triaged to trauma centers. The number of more seriously injured patients, as estimated by length of hospital stay of more than four days; Injury Severity Score of more than 15; fatality; or patients requiring surgery within six hours for trauma to the chest, abdomen, central nervous system, and/or major vessel, also increased. The rates of increase were more than those of the population (15%) for the Injury Severity Score of more than 15 group (79%) and the length of stay of more than four days group (108%), but lower for the fatality group (11%) and the early surgery group (7%). Because traffic and motor vehicle accidents comprise 70% to 80% of all trauma injuries, a separate analysis was conducted for these cases, using numbers of miles traveled as a basis for analysis. The analysis revealed a significant (alpha = .05) increase in over-triage rate, from 43% in 1981 to 74% in 1988, for motor vehicle accident patients. No changes in trends were found in the pre-analysis and postanalysis. Changes in trends occurred in 1986. CONCLUSION: The growth rate for the total trauma population exceeds that for overall county population growth. These results suggest overtriage. No changes in trends were found after the 1983 revisions in triage criteria. Changes in trends were found in 1986 after the revisions in the Injury Severity Score scoring system.

California↗
PubMed1992

Effects of adrenoceptor blockade on cardiac hypertrophy and myocardial phospholipids.

Catecholamines have been proposed as a stimulus for the hypertrophic response to pressure overload of the heart and could also mediate the membrane lipid changes associated with cardiac hypertrophy. To address both of these possibilities, cardiac hypertrophy was induced by aortic constriction in the presence or absence of chronic alpha- or beta-adrenoceptor blockade. Heart weights and heart weight to body weight ratios in aortic-constricted rats of the adrenoceptor-blocked and vehicle-treated groups were elevated to the same extent when compared with values in sham-operated rats of each group. Analysis of the fatty acyl composition of the major phospholipid classes revealed that similar changes occurred in vehicle-treated, alpha-blocked, and beta-blocked aortic-constricted rats when compared with respective groups of sham-operated rats. Specifically, linoleic acid was reduced in the phosphatidylcholine, phosphatidylethanolamine (PE), and cardiolipin (CL) fractions in all groups of aortic-constricted rats. This reduction was accompanied by increased docosahexaenoic acid, arachidonic acid, or palmitic acid in phosphatidylcholine; docosahexaenoic acid in phosphatidylethanolamine; and oleic acid in cardiolipin fractions. Adrenoceptor blockade did not prevent or attenuate the major changes in the fatty acyl composition of phospholipids or the increase in heart weight associated with aortic constriction. This suggests that a change in the level of adrenoceptor stimulation is not the stimulus for cardiac hypertrophy or the observed alterations in phospholipid composition in the pressure-overloaded rat heart.

Adrenergic alpha-Antagonists↗
PubMed1991

Flash photolysis of magnesium-DM-nitrophen in heart cells. A novel approach to probe magnesium- and ATP-dependent regulation of calcium channels.

Changes in cytoplasmic magnesium concentration in the millimolar range are known to evoke directionally opposite changes in the amplitude of cardiac L-type calcium current, but the effects of magnesium changes in the micromolar range are not known. This concentration range is particularly interesting for two reasons: open-channel block of some ion channels (eg, the inward rectifier) occurs with a micromolar Kd; and magnesium at such low levels may limit the availability of magnesium adenosine triphosphate (MgATP) and magnesium guanidine triphosphate (MgGTP) for a variety of enzymatic processes, including calcium channel phosphorylation. To investigate the effects of critical magnesium depletion and its sudden reversal, we used flash photolysis to liberate magnesium from intracellular DM-nitrophen in guinea pig heart cells. Under our conditions, intracellular [Mg2+] changed from 0.25 mumol/L before the flash to less than or equal to 200 mumols/L afterwards. Calcium channel currents carried by either calcium or barium consistently increased after flash, a directional change opposite to the reported effects of elevated [Mg2+] in the millimolar range. Our data do not yet enable us to understand the basis of the magnesium effect, but we are particularly interested in the possibility that the sudden appearance of magnesium enables calcium channel phosphorylation.

Acetates↗
PubMed1990

High-speed digital imaging of cytosolic Ca2+ and contraction in single cardiomyocytes.

A charge-coupled device (CCD) camera, with the capacity for simultaneous spatially resolved photon counting and rapid frame transfer, was utilized for high-speed digital image collection from an inverted epifluorescence microscope. The unique properties of the CCD detector were applied to an analysis of cell shortening and the Ca2+ transient from fluorescence images of fura-2-loaded [corrected] cardiomyocytes. On electrical stimulation of the cell, a series of sequential subimages was collected and used to create images of Ca2+ within the cell during contraction. The high photosensitivity of the camera, combined with a detector-based frame storage technique, permitted collection of fluorescence images 10 ms apart. This rate of image collection was sufficient to resolve the rapid events of contraction, e.g., the upstroke of the Ca2+ transient (less than 40 ms) and the time to peak shortening (less than 80 ms). The technique was used to examine the effects of beta-adrenoceptor activation, fura-2 load, and stimulus frequency on cytosolic Ca2+ transients and contractions of single cardiomyocytes. beta-Adrenoceptor stimulation resulted in pronounced increases in peak Ca2+, maximal rates of rise and decay of Ca2+, extent of shortening, and maximal velocities of shortening and relaxation. Raising the intracellular load of fura-2 had little effect on the rising phase of Ca2+ or the extent of shortening but extended the duration of the Ca2+ transient and contraction. In related experiments utilizing differential-interference contrast microscopy, the same technique was applied to visualize sarcomere dynamics in contracting cells. This newly developed technique is a versatile tool for analyzing the Ca2+ transient and mechanical events in studies of excitation-contraction coupling in cardiomyocytes.

Animals↗
PubMed1987

Mechanisms for altered carnitine content in hypertrophied rat hearts.

Carnitine levels are reduced in hypertrophied hearts of rats subjected to aortic constriction (banding) and elevated in hypertrophied hearts of spontaneously hypertensive rats (SHR). In an attempt to determine the mechanisms for these alterations, L-[14C]carnitine transport was examined in isolated perfused hearts. Total carnitine uptake was significantly reduced by approximately 20% in hypertrophied hearts of banded rats at all perfusate carnitine concentrations employed. The reduction in total uptake was due to a 40% reduction in carrier-mediated carnitine uptake with no difference in uptake by diffusion. In contrast, carnitine uptake was not altered in isolated hypertrophied hearts of SHR. However, serum carnitine levels were elevated in SHR, which could result in increased myocardial carnitine uptake in vivo. The data suggest that altered carnitine content in hypertrophied hearts of aortic-banded rats is due to an alteration in the carrier-mediated carnitine transport system in the myocardium. However, altered carnitine content in hypertrophied hearts of SHR is not due to a change in the carnitine transport system per se but may rather be due to a change in serum carnitine levels.

Animals↗